The lymphoid system is composed of many different cell types
distributed in various tissues, but it functions as a single entity.
This cohesion is possible primarily because immune cells are mobile;
among them, large numbers of lymphocytes traffic continually in a highly
regulated manner between secondary lymphoid tissues, where antigens,
antigen-presenting cells, immunocompetent lymphocytes, and regulatory
cells are located. (1-3)

Under physiological conditions, naive (i.e., mature, non-activated)
lymphocytes migrate from the peripheral blood into lymph nodes and
Peyer's patches by selectively interacting with the specialized
endothelium of postcapillary venules called high endothelial venules
(HEVs). HEVs are distinctive microvascular segments located mainly in
the interfollicular area of these tissues and consist of tall and
cuboidal endothelial cells (ECs) surrounded by a thick basal lamina and
a prominent perivascular sheath. A remarkable feature of HEVs is that
their ECs allow numerous lymphocytes to adhere to and migrate between EC
junctions, during which both cell types display markedly motile changes.
(4)

Flat-walled venules in non-lymphoid tissues including the skin also
support leukocyte extravasation under physiological conditions, but
unlike HEVs, they allow mainly memory-type lymphocytes and dendritic
cells (DCs) to extravasate, (1-3) and the extent of extravasation is
much smaller than that observed with HEVs. Relatively little is known
about the molecular mechanisms underlying leukocyte extravasation
through the flat-walled venules, although both adhesion molecules and
chemokines appear to play important roles, as is the case with HEVs.

2. Molecules regulating lymphocyte migration across blood vessels

A) HEVs in peripheral lymph nodes. In the early 1980s, Yamaguchi
and Schoefl documented that circulating lymphocytes are able to
selectively recognize and adhere to the lumen of HEVs and that this
mechanism is influenced by the circulating lymphocyte level. (5) They
found through meticulous electron microscopic analyses that, whereas
about 40% of the lymphocytes interacting with the HEV ECs are in the
process of transmigrating in normal mice, about 90% of the interacting
cells are in the process of transmigrating in lymphopenic mice in which
circulating lymphocytes were depleted by thoracic duct cannulation, (5)
indicating that lymphocyte transmigration is upregulated when the
circulating lymphocyte level is low. They also found in the
lymphocyte-depleted mice that intravenously injected lymphocytes swiftly
adhere to HEVs and penetrate the HEV wall (Fig. 1) and that the speed
and intensity of lymphocyte binding/transmigration are both greatly
enhanced compared with normal mice. (5) These observations indicated
that lymphocyte trafficking across HEVs is homeostatically regulated by
the number of lymphocytes in the blood. It could be that a humoral
factor(s) produced under lymphopenic conditions acts on lymphocytes
and/or HEV ECs to enhance lymphocyte transmigration.

It was subsequently found that the lymphocytes' interaction
with HEV ECs is directed by a sitespecific adhesion cascade involving
several specific molecules and chemokines that act in sequence (Fig. 2).
(1,2,6) This adhesion cascade is initiated by leukocyte
tethering/rolling, followed by the firm arrest of rolling lymphocytes,
and finally by the transvenular migration (extravasation) of
lymphocytes. (2,3,7) This entire process is very similar to that
observed in the lumen of inflamed blood vessels, (8) although some
distinct and specific molecules are used in HEVs, as detailed below.

i) Tethering/Rolling. As naive lymphocytes flow into HEVs, they
decelerate rapidly and exhibit weak, transient, on-and-off adhesive
interactions (tethering) with the HEV ECs and roll along the inner
surface of the HEV wall (rolling). This process is mediated by CD62L
(L-selectin) on lymphocytes and by sialomucins expressed on HEV ECs
(Fig. 2). L-selectin is a lectin-type cell adhesion molecule that
recognizes sugars and is expressed on all leukocytes. It binds to
specific O-glycans expressed on HEV sialomucins, a group of heavily
glycosylated proteins (mucins) whose carbohydrate moieties contain
sialic acid. The critical recognition determinant on the O-glycans is
6-sulfo sialyl Lewis X ([sLe.sup.x]), which serves as a capping
structure on core-2 and extended core-1 branches, and is specifically
recognized by the MECA-79 monoclonal antibody. HEVs in the peripheral
lymph nodes express at least five different flat endothelial cells in
cutaneos venules sialomucins, including GlyCAM-1, (9) CD34, (10)
podocalyxin, (11) endomucin, (12) and nepmucin/CD300g. (13) To
synthesize the L-selectin-binding MEC-79reactive [sLe.sup.x] structures,
HEV ECs express a set of glycosyltransferases, including
[alpha]1,3-fucosyltransferases IV and VII, Core1-[beta]3GlcNAcT (also
known as [beta]3GlcNAcT-3 or Core1-GlcNAcT), Core2-[beta]1,6GlcNAcT
(Core2-GlcNAcT), GlcNAc6ST-1, and GlcNAc6ST-2. (14)

The HEV sialomucins are also called peripheral node addressins
(PNAds), because they function as "address code" molecules for
peripheral node HEVs. These sialomucins share a common sugar epitope,
i.e., the 6-sulfo [sLe.sup.x] structure mentioned above.

Binding of the MECA-79 antibody to this epitope abrogates
L-selectin-PNAd interactions. In mesenteric lymph node HEVs, MAdCAM-1, a
sialomucin bearing two immunoglobulin-like domains, (15) serves as a
vascular addressin, and [alpha]4[beta]7 integrin serves as the cognate
lymphocyte receptor that mediates rolling and adhesion, (16) as
described below. Within the HEV lumen, all L-selectin-expressing
leukocytes undergo rolling/tethering, but as described below, only
lymphocytes show firm adhesion to HEV ECs (2) (Fig. 2).

ii) Firm arrest/ adhesion. After tethering/ rolling, lymphocytes
further decelerate and undergo a shear-resistant firm arrest/adhesion to
the HEV wall, which is primarily mediated by an interaction between the
[beta]2 integrin LFA-1 (CD11a/CD18) on lymphocytes and ICAM-1/ICAM-2 on
HEV ECs (Fig. 2). While L-selectin is constitutively active, integrins
generally need to be activated to mediate adhesion. This integrin
activation is induced by Gprotein coupled receptor (GPCR) signaling in
the lymphocytes at HEVs, which is initiated by the lymphocytes'
interaction with chemokines displayed on ECs via specific receptors.
Chemokines are generally positively charged, and hence bind to
negatively charged molecules such as certain glycosaminoglycan chains
that are abundantly expressed on HEV ECs; this binding prevents the
chemokines from getting washed off by the blood flow. (2,17) In the
peripheral lymph nodes, LFA-1 on T cells is activated when
HEV-associated chemokines including CCL21/ CCL19 and CXCL12 interact
with their specific receptors on T cells, CCR7 and CXCR4, respectively,
whereas LFA-1 on B cells is activated by interactions with CXCL13 via
CXCR5. Because these chemokine receptors are expressed preferentially on
lymphocytes, only lymphocytes exhibit firm adhesion via activated LFA-1,
which then binds to the immunoglobulin-like-domain-containing adhesion
molecules, ICAM-1 and ICAM-2 on the surface of HEV ECs.

iii) Intraluminal crawling and transmigration. Upon undergoing firm
adhesion, naive lymphocytes start to crawl along the luminal surface of
HEVs (intraluminal crawling) and slowly migrate to distant emigration
sites, where they then transmigrate at certain hot spots ("exit
ramps") (4) along the HEV wall (Fig. 2). The HEV basal lamina has
numerous pores, through which lymphocytes pass to reach the abluminal
side of HEVs, without disrupting the basal lamina. The basal lamina
consists of type IV collagen, fibronectin, and laminin, which allow
chemokine immobilization mainly via electrostatic interactions. Hence,
the HEV basal lamina binds locally produced lymphoid chemokines,
including CCL21, CCL19, CXCL12, and CXCL13, creating a chemokine-rich
environment. The HEV basal lamina also functions as a guidance structure
for the directional trafficking of lymphocytes from HEVs into the
lymphoid tissue parenchyma. (2)

Although lymphocytes were reported to cross the endothelial barrier
using both paracellular (between adjacent ECs) and transcellular
(through the cytoplasm of ECs) migration routes, Schoefl clearly showed,
using electron microscopic and mathematical analyses, that lymphocytes
predominantly use the paracellular route to exit HEVs. (18)

In addition to the mechanisms described above, a variety of other
adhesion molecules expressed on HEV ECs have been implicated in
lymphocyte transmigration, although their modes of action remain ill
defined. These molecules include CD31, VCAM-1, JAM-A, JAM-B, JAM-C,
ESAM, VEcadherin, and nepmucin (CD300g). (19)

A specific lysophospholipid, lysophosphatidic acid (LPA) plays a
particularly important role in the lymphocyte transmigration across
HEVs. LPA is a naturally occurring bioactive lysophospholipid,
consisting of a phosphate, a glycerol, and a fatty acid (Fig. 3a). LPA
is mainly derived from its precursor, lysophosphatidylcholine, which is
abundant in plasma, by the enzymatic action of the lysophospholipase D,
autotaxin (ATX). When LPA binds to one of its specific receptors,
[LPA.sub.1] -[LPA.sub.6], which are all GPCRs, multiple signaling
pathways are activated with various downstream physiological and
pathological effects. Approximately ten years ago, we (20) and Steve
Rosen's group (21) found that the primary LPA-producing enzyme ATX
is transcribed abundantly in HEV ECs. Subsequent analyses showed that
the ATX expressed in HEVs regulates lymphocyte trafficking by locally
generating LPA. Local inhibition of the ATX/LPA axis substantially
blocks the lymphocyte transmigration across HEVs, while a local
administration of LPA abrogates this effect. (22) At the HEV lumen, LPA
acts directly on HEV ECs via its receptors [LPA.sub.4] and [LPA.sub.6],
although its actions through these receptors are different, given that
LPA (4) deficiency causes delayed lymphocyte transmigration across the
HEV wall, whereas [LPA.sub.6] deficiency also compromises lymphocyte
transmigration but to a much lesser extent. (23) In an in vitro
transmigration assay, ATX inhibition impairs the release of lymphocytes
that migrate underneath HEV ECs, and this defect is abrogated by adding
LPA; LPA appears to contribute to lymphocyte de-adhesion (or release)
from ECs by regulating the myosin II activity in HEV ECs (Fig. 3b). (22)

iv) Migration of non-lymphoid cells across HEVs. Under
physiological conditions, not only naive lymphocytes but also DC
precursors, plasmacytoid DCs (pDCs), and central memory T cells
extravasate from HEVs. While the DC precursor migration into lymph nodes
is thought to follow basically the same steps as that of naive
lymphocytes, (24) its precise mechanism remains to be fully explored.
The pDCs show robust transmigration underneath HEV ECs but not non-HEV
ECs, using adhesion molecules very similar to those used by naive
lymphocytes. (25) The pDCs also require CCR7 to enter the lymph nodes
via HEVs. (26,27) This is also the case for central memory T cells,
which readily proliferate and differentiate into effector cells in
response to their antigenic stimulation in lymph nodes. These cells
character istically express high levels of L-selectin and CCR7, which
they use to interact with HEV ECs, just like naive T cells do. However,
to what extent these cells require the HEV-associated lysophospholipid
LPA for their transmigration remains to be explored.

When sterile inflammation occurs locally, bloodborne neutrophils
rapidly and abundantly migrate into the draining lymph nodes via HEVs.
Under these conditions, IL-17-producing lymphocytes first migrate into
the draining lymph nodes, where they produce IL-17, which induces the
production of CXCL2, a chemokine ligand for CXCR2, in HEVs, leading to
the HEV-mediated migration of CXCR2expressing neutrophils from the blood
into the draining lymph nodes. The effect of IL-17 on CXCL2 depends on
IL-1[beta], which is also enhanced by IL-17. (28) Thus, although
neutrophils are prevented from entering lymph nodes via HEVs under
physiological conditions, they can migrate into lymph nodes abundantly
when HEVs undergo an inflammation-induced molecular switch, which
initiates the neutrophils' CXCR2 engagement by CXCL2 displayed on
ECs.

B) HEVs in intestinal lymphoid tissues. The lymphocyte trafficking
to the small intestine is governed by two types of adhesion pathways.
One is mediated by the interaction between lymphocyte L-selectin and
HEV-expressed sialomucins/PNAds, which is mainly used by naive
lymphocytes, and the other is mediated by the interaction between
lymphocyte integrin [alpha]4[beta]7 and the vascular cell adhesion
molecule MAdCAM-1, which is mainly used by lymphocytes that have been
exposed to antigen-experienced DCs in the small intestine. When naive
lymphocytes that have migrated into the small intestine encounter DCs,
they are exposed to high concentrations of DC-derived retinoic acid and
start to upregulate their expressions of the integrin [alpha]4[beta]7
and the chemokine receptor CCR9. (29) The [alpha]4[beta]7 specifically
binds MAdCAM-1, and CCR9 is the receptor for the chemokine CCL25
secreted by small intestinal venules. Thus, these lymphocytes use
[alpha]4[beta]7 and CCR9 to recognize tissue-specific cues expressed on
small intestinal ECs, i.e., MAdCAM-1 and CCL25. Recently, the orphan
chemokine receptor GPR15 has been shown to control the localization of T
effector cells in the colon. (30)

C) Flat ECs in peripheral tissues. Flat ECs found in
non-specialized regular postcapillary venules in the skin also mediate
constitutive immune cell trafficking under steady state conditions,
albeit to a much lesser extent than do HEV ECs. These ECs support
leukocyte rolling under non-inflamed conditions, (31) due to a
constitutive, low-level expression of E- and P-selectins. The rolling
frequency is largely determined by P-selectin, with E-selectin playing a
smaller role, and L-selectin is not involved. (32) Most skin T cells
express an E- and P-selectin--binding molecule, the cutaneous lymphocyte
antigen (CLA), which is derived from the glycosylation of a lymphocyte
sialomucin PSGL-1, and a chemokine receptor CCR8, whose expression is
induced by keratinocyte-derived factors. (33) Upon binding to
EC-displayed selectins, the CLA-expressing T cells extravasate from
dermal venules. The engagement of lymphocyte CCR8 with its ligand CCL1
constitutively expressed in the dermal venules is thought to promote
this extravasation by activating lymphocyte integrins. Skin T cells also
express another chemokine receptor CCR4, whose engagement with a dermal
venule-expressed chemokine, CCL17, promotes T cell migration into the
skin. In inflamed skin, activated T cells express high levels of CCR10,
whose engagement with CCL27, which is produced by keratinocytes and is
highly displayed on inflamed venules in the skin, is critical for T cell
recruitment to the skin. (34) Interestingly, skin DCs are able to
produce the active vitamin D3 metabolite 1,25[(OH).sub.2]D3 from
sunlightinduced vitamin D3, and 1,25[(OH).sub.2]D3 upregulates the CCR10
expression in T cells. (35) However, CCR10 is largely absent from the T
cells in uninflamed skin; (33,36) hence, the CCL17/CCR10 axis appears to
be important for T cell migration into inflamed skin but not for the
constitutive T cell migration into normal skin.

Recent studies indicate that T cells are abundant in the skin and
have unique immunological abilities. (37) A careful study in humans
indicated that 2 x [10.sup.10] T cells exist in the skin, which is
almost twice the number of T cells in the entire circulation. (38) Most
of the skin T cells have the phenotype of effector memory T cells, and
some are central memory T cells. The prevailing hypothesis is that
effector memory T cells that arise upon the antigenic stimulation of
naive as well as central memory T cells in lymph nodes migrate into
peripheral tissues via venules bearing flat ECs and return to the lymph
nodes via lymphatics, whereas the central memory T cells that also arise
in lymph nodes recirculate between the blood vascular and lymphatic
vascular systems using HEVs, just like naive T cells. (39,40) However,
the presence of CLA-expressing central memory
([L-selectin.sup.+][CCR7.sup.+]) T cells in the skin (~20% of normal
skin T cells) in humans (41) indicates that not all of the central
memory T cells recirculate via the conventional route; some of them
migrate into the periphery and then move to the lymph nodes via
lymphatics. On the other hand, a substantial proportion of memory T
cells in the skin appear to be sessile and do not leave the tissue;
hence, they are now called resident memory T cells ([T.sub.rM]s). (42)
These cells provide effective protection against local antigen
re-challenge. The failure of these cells to exit the tissue is currently
thought to be due to their low expression of the transcription factor
KLF2 and of S1PR1 and to their high expression of the C-type lectin
CD69; as a result of these conditions, the cells fail to respond to an
"exit cue" provided by S1P (sphingosine-1-phosphate), which is
released mainly from lymphatic ECs. S1P's involvement in lymphocyte
egress will be discussed below.

Regulatory T cells (Tregs) are also found in the skin, where they
comprise 10~20% of the normal skin T cells in man (38) and mouse. (43)
They constitutively migrate to the draining lymph nodes via lymphatics
in the steady state. (44) These cells show increased migration during
cutaneous immune responses and return to the skin upon re-exposure to
antigen. The migrating Tregs have a stronger immunosuppressive effect
than lymph node-residing Tregs and appear to contribute to the
downregulation of cutaneous immune responses. (44) These Tregs express
CD103, CCR4, and CCR5, but the molecular mechanism underlying their
migration from the skin to lymph nodes remains unclear.

A recent study using transgenic mice expressing a photoconvertible
fluorescent protein, Kaede, confirmed that DCs also continuously migrate
from the skin to draining lymph nodes under steady-state conditions.
(44) DCs do not apparently require [beta]2 integrins for their
migration, since CD18-/- mice deficient in the [beta]2 integrin subunit
show uncompromised DC migration from the blood to normal or inflamed
skin, or from the skin to draining lymph nodes. (45) This finding was
later verified by multiphoton microscopy, which showed that DCs crawl
into lymphatics independent of integrins (46) and that they are guided
into lymphatics by a tissue-immobilized gradient of CCL21 in an
integrin-independent but CCR7-dependent manner. (47)

Innate lymphoid cells (ILCs) are a group of non-T, non-B
lymphocytes that are important in innate immune responses and in the
regulation of inflammation. (48) In the skin, group 2 ILCs are
relatively abundant and continuously patrol the tissue at speeds
comparable to those described for dermal DCs, frequently interacting
with perivascular mast cells. (49) However, how these cells are
recruited to the skin and whether they migrate from the skin into the
draining lymph nodes remain unknown.

3. Molecules regulating DC migration into lymphatics

a) Chemokines and their receptors. The trafficking of lymphocytes
and DCs into the lymphatic vessels is an active process. (50) Chemokines
presented on lymphatic ECs are important for attracting and guiding DCs
into lymphatics, and DCs interacting with the lymphatic ECs respond to
these chemokines. For instance, a CCR7-ligand chemokine, CCL21, is
abundantly expressed on lymphatic capillaries and induces the chemotaxis
of CCR7-expressing DCs, allowing them to migrate toward and to enter
into lymphatics. CCL21 binds a lymphatic EC marker podoplanin with high
affinity, is expressed on the basal lamina of lymphatics, and is shed
into the perivascular stroma, (51) which may contribute to the formation
of a peri-lymphatic CCL21 concentration gradient. In addition, a
chemokine-scavenging molecule, CCRL1, is expressed by lymphatic ECs that
line the ceiling but not the floor of the subcapsular sinus. (52) CCRL1
sequesters and induces CCL21 degradation, which is thought to contribute
to the formation of a CCL21 concentration gradient from the sinus toward
the lymph node parenchyma, which helps direct DCs to migrate toward the
inner areas of lymph nodes. Indeed, DCs have been shown to require the
lymphatic ECdisplayed CCL21 to enter the lumen of lymphatics, in a
CCR7-dependent manner. (54,54)

In addition, CCR7's function can be modulated locally; CCR7 is
upregulated by locally generated molecules including prostaglandin E2
(55) and extracellular [NAD.sup.+], (56) both of which are released from
damaged or inflamed cells. Notably, however, while DC migration across
the subcapsular sinus lymphatic EC layer is CCR7-dependent, T cell
migration across the sinus lymphatic ECs is completely independent of
CCR7 signaling. (57) Thus, the CCL21-CCR7 axis does not appear to be the
only regulator of immune cell trafficking across the lymph node
subcapsular sinus.

Under inflamed conditions, the chemokine CX3CL1 (fractalkine)
appears on lymphatic ECs and is actively secreted, and the soluble
rather than membrane-anchored chemokine promotes DC migration toward
lymphatics and EC transmigration. (58) CXCL12 is also induced on the
surface of lymphatic ECs upon an inflammatory stimulus, and DCs migrate
across these cells in a CXCL12/CXCR4dependent manner. (59) In the case
of lymphocytes, CXCL12 acts in synergy with CCR7 ligands to promote cell
migration by sensitizing the cells through CXCR4, thus enabling them to
respond to lower concentrations of CCR7 ligands. (60) Given that mature
DCs also express both CCR7 and CXCR4 at levels comparable to those on
lymphocytes, chemokine-induced synergy may also enhance DC recruitment
into lymphatics under certain conditions.

Interestingly, CCL1 is expressed by the subcapsular sinus lymphatic
ECs of skin-draining lymph nodes but not by capillary lymphatic vessels
in the skin, and inhibiting the CCL1/CCR8 interaction leads to impaired
DC migration into the lymph node parenchyma, indicating that the
CCL1/CCR8 axis functions downstream of the DC entry into lymphatics by
regulating entry into the subcapsular sinus of the lymph nodes. (61)
Notably, malignant melanoma cells often express high levels of CCR8 and
use CCL1-CCR8 to enter into the lymph node parenchyma, forming lymph
node metastases. (62) Thus, certain types of tumor cells can coopt the
normal mechanism of CCL1-CCR8-dependent immune cell trafficking to
metastasize to the lymph node.

b) Adhesion molecules and their receptors.

As mentioned above, integrins are not required for the DC migration
into afferent lymphatics in the steady state. (46) Podoplanin expressed
on lymphatic ECs can capture CCL21 (see above) and also binds a
lectin-type molecule CLEC-2. Podoplanin's binding to CLEC-2
promotes the formation of actin-rich protrusions on DCs, which allow the
DCs to spread along stromal scaffolds and support DC motility. (63)
While integrin-binding molecules including ICAM-1 and VCAM-1 are
expressed at only low levels in lymphatic ECs, their expression is
strongly upregulated during inflammation, and they contribute to DC
migration by interacting with [beta]2 integrins (LFA-1 and Mac-1) (64)
and [beta]1 integrin VLA-4, (65) respectively.

Lymphatic ECs produce an immunomodulatory molecule, semaphorin 3A.
This molecule promotes actomyosin contraction in the trailing edge of
DCs by binding plexin A1 on DCs and may induce the disassembly of
adhesive components at the trailing edge as well, thus promoting DC
transmigration. (66)

4. Lymphocyte migration across the lymphoid tissue parenchyma

Once lymphocytes have entered the lymph nodes, they search for
antigen by moving in the cell-dense three-dimensional network in the
parenchyma, a highly confined environment. Katakai et al. (67) reported
that ATX produced by lymph node stromal cells promotes
integrin-independent, Rho-dependent interstitial T cell motility in
lymph nodes. Our group subsequently reported (68) that ATX produced by
fibroblastic reticular cells (FRCs) generates LPA through its enzymatic
activity and that LPA enhances lymphocyte motility through the densely
packed lymph node reticular network (Fig. 3c). Imaging mass spectrometry
analysis showed that biologically relevant LPA species (LPA 18:1, 18:2,
and 20:4) are expressed in the lymph node paracortex at sites that are
either close to or distant from HEVs and that the latter is selectively
reduced in mice that are ATX deficient specifically in FRCs, (68) in
agreement with the hypothesis that FRCs produce LPA by the ATX on their
cell surface. Furthermore, intravital two-photon microscopic analysis
showed that T cell migration in the parenchyma is significantly
attenuated in the conditional ATX-deficient mice compared to control
mice and that the ATX/LPA-dependent T cell motility is mediated by the
LPA receptor [LPA.sub.2] on the T cell surface. (68) These results,
together with the finding that LPA activates Rho-ROCK-myosin II pathways
via LPA2 in T cells, suggest that the LPA generated by FRCs acts locally
on T cells via LPA2, thereby regulating T cell contractility and
motility in the lymph node reticular network (68) (Fig. 3c).

5. Lymphocyte egress from lymphoid tissues

Lymphocyte egress from lymphoid tissues is currently thought to be
regulated primarily by sphingosine-1-phosphate (S1P), which is
structurally similar to LPA. S1P acts on a family of five
G-protein-coupled receptors ([S1P.sub.1]--[S1P.sub.5]) and is rapidly
degraded into a biologically inactive form by S1P phosphatases and S1P
lyase in vivo.

S1P is released from erythrocytes and vascular ECs, causing a high
S1P concentration in the peripheral blood. In contrast, S1P is
relatively scarce in lymphoid tissues, due to an abundance of S1P lyase.
This differential concentration of S1P between the blood and lymphoid
tissue is currently thought to drive lymphocyte emigration from lymphoid
tissues. Lymphocytes within the lymphoid tissues express high levels of
the S1P receptor [S1P.sub.1] and thus undergo chemotaxis in response to
the S1P gradient. In contrast, peripheral blood lymphocytes express low
levels of [S1P.sub.1], due to its downregulation by internalization in
response to the high S1P concentration in the blood. When blood-borne
lymphocytes enter lymphoid tissues, [S1P.sub.1] is upregulated due to
the paucity of S1P in the tissue. Within the lymph nodes, the
lymphocytes are then transported to the cortical sinuses, the medullary
sinus, and finally to the efferent lymphatics, by sensing the S1P
concentration gradient in an [S1P.sub.1]-dependent manner. This cyclical
change in lymphocyte [S1P.sub.1] expression, which has been proposed to
direct lymphocyte egress from the lymph nodes, (69) may similarly
regulate lymphocyte egress from the thymus.

However, this widely accepted hypothesis cannot be readily
reconciled with the following findings. First, S1P1 transcripts are also
abundant in the ECs and vascular smooth muscle cells surrounding blood
vessels, and strong [S1P.sub.1] activation is detected in both the
lymphatic and vascular ECs in lymphoid tissues, where most lymphocytes
show no evidence of [S1P.sub.1] activation under homeostatic conditions.
(70) Second, [S1P.sub.1] is also expressed at high levels on
macrophages, DCs, and natural killer cells, but only lymphocytes exit
the lymph nodes in response to physiological concentrations of S1P.
These findings indicate that S1P's role in regulating lymphocyte
egress is more complex than described so far.

S1P also appears to regulate the barrier function of the HEVs in
antigen-stimulated lymph nodes. Herzog et al. (71) reported that
platelets migrate across HEVs together with lymphocytes in mesenteric
lymph nodes and are activated by specific interactions between the
platelet cell surface lectin CLEC-2 and podoplanin, expressed on the
surrounding FRCs. The activated platelets then secrete S1P, which
stimulates the HEVs to maintain their vascular integrity. However, the
effect of the podoplaninCLEC-2 interaction on HEV integrity can only be
detected in mesenteric lymph nodes, where exogenous antigens are
abundant, and not in peripheral lymph nodes unless the mice are
immunized, implying that this mechanism is important under inflammatory
conditions.

6. Concluding remarks

In the past decade, we have learned many details about the
molecular mechanisms underlying immune cell migration across blood and
lymphatic vessels. However, many questions and challenges still remain.
For instance, we still do not know what promotes the generation of HEVs
in tissues. This information is important for devising novel therapeutic
approaches for autoimmune diseases and cancer, because by increasing or
decreasing HEVs, one should be able to control the lymphocyte
recruitment into tissues, and thus the immune status of particular
tissues. We also do not know what regulates the permeability of HEVs;
for example, whether increased HEV permeability is an intrinsic property
of ECs or of other cells surrounding the HEVs, or whether
lysophospholipids including LPA and S1P are involved in this phenomenon.
This knowledge would also be relevant for the artificial manipulation of
immune responses. Given that it is now clear that both blood vascular
and lymphatic vascular ECs are the critical regulators of immune cell
trafficking, elucidating the above points will bring new insight into
the host defense mechanisms and pathogenesis of inflammatory disease,
and will provide valuable information for the design of anti-migration
therapies for inflammation.

doi: 10.2183/pjab.93.012

Acknowledgements

We are grateful to Dr. Kazuhito Yamaguchi for allowing us to use
his splendid SEM photographs of HEVs. We also thank Prof. Tamio Yamakawa
for providing us the opportunity to compile this manuscript. One of the
authors (MM) wishes to thank his mentors, Drs. Masahiko Kotani, Peter
McCullagh, and Zdenek Trnka for their wisdom, guidance, and support
throughout the years. Masahiko Kotani introduced MM to the subject of
lymphocyte recirculation and has deeply inspired his research ever
since. Peter McCullagh encouraged MM to pursue his research endeavors.
His sincere enthusiasm for science sets the standard to which MM seeks
to aspire. MM is grateful to Zdenek Trnka for many inspiring
conversations and helpful suggestions during his tenure in Basel,
Switzerland. The authors acknowledge all the members of the Laboratory
of Immunodynamics, the Graduate School of Medicine, Osaka University,
and our collaborators, who actively participated in the experiments
discussed herein. Because of space limitations, we were able to cite
only a portion of the relevant literature, and we apologize to
colleagues whose contributions might not be appropriately acknowledged
in this review.

Masayuki Miyasaka was born in 1947 in Ueda, Nagano Prefecture. He
is Professor Emeritus of Osaka University, Japan, and a FiDiPro (Finland
Distinguished Professor) of the Academy of Finland. He was formerly
Professor and Chairman of the Laboratory of Immunodynamics, Department
of Immunology and Microbiology at the Osaka University Graduate School
of Medicine in Osaka, Japan (1994-2012). He was President of the
Japanese Society for Immunology (2006-2008). He was also an Editor of
FEBS Letters (1998-2012), an Associate Editor of Immunology (2004-2007),
and an Associate Editor of International Immunology (1989-2017). He
received the M.D. from the Kyoto University School of Medicine in Japan
in 1973 and Ph.D. in immunology from the John Curtin School of Medical
Research, Australian National University in Canberra in 1981. He then
served as a member of the Basel Institute of Immunology in Switzerland
(1981-1986), where he studied the ontogeny of the lymphoid system and
lymphocyte migration. Currently, Dr. Miyasaka is interested in the
molecular mechanisms underlying lymphocyte trafficking into various
tissues and also the mechanism of tumor metastasis in vivo. Main topics
of his research are 1) physiological recruitment of lymphocytes and
dendritic cells from the body into secondary lymphoid tissues and 2)
functions and regulation of intercellular adhesion molecules and
chemokines, and their regulators.

Caption: Fig. 1. Lymphocyte transmigration across HEVs is regulated
homeostatically. Left; Scanning electron micrograph (SEM) of a sectioned
HEV of a normal mouse, showing numerous lymphocytes adhering to
endothelial cells before migrating between them (Courtesy of Dr. K.
Yamaguchi, Yamaguchi University School of Medicine). Three of them
(indicated by asterisk) are in the process of transmigrating, as judged
by their reduced cell size and microvilli. Right; SEM of a sectioned HEV
of a mouse that was subjected to lymphocyte depletion by chronic
thoracic duct cannulation and then injected with lymphocytes
intravenously. Scale bar: 10 pm. The injected lymphocytes quickly
adhered to the endothelial surface and started penetrating the
endothelial wall; all of the cells in the field are in the process of
ransmigrating (a transmigrating lymphocyte at the top left is magnified
in the inset at the bottom right). These observations show that
lymphocyte transmigration is markedly upregulated when the circulating
lymphocyte level is low.

Caption: Fig. 2. The lymphocyte adhesion cascade in the lumen of
HEVs and cutaneous venules. In peripheral lymph node HEVs, lymphocytes
initiate rolling through L-selectin--sialomucin interactions.
Subsequently, the LFA-1 integrin on T cells is activated mainly by
chemokines (CCL21 and CCL19). The T cells then bind to EC molecules such
as ICAM-1 through the activated LFA-1, and proceed to migrate across
HEVs. In Peyer's patch HEVs, lymphocyte rolling is mediated by L-
selectin--sialomucin interactions and [alpha]4[beta]7 integrin--MAdCAM-1
interactions. The chemokines that trigger integrin activation in T and B
cells appear to be the same as in lymph nodes. In cutaneous
post-capillary venules, T cell rolling, integrin activation and cell
adhesion are regulated by PSGL-1-selectins interactions, chemokines
including CCL1 and CCL17, and LFA1-ICAM-1 interactions, respectively.

Caption: Fig. 3. A specific lysophospholipid, LPA, promotes
lymphocyte extravasation from HEVs and lymphocyte motility in the lymph
node parenchyma. (a) Structure of LPA. LPA has a phosphate group, a
glycerol backbone, and a single fatty acid chain. (b) In the vicinity of
HEVs, ATX is expressed at high levels and generates LPA locally. LPA in
turn acts on HEV ECs via [LPA.sub.4] and [LPA.sub.6] to increase their
motility, promoting dynamic lymphocyte-HEV interactions and subsequent
lymphocyte de-adhesion from HEV ECs at the steady state. (c) In the
lymph node cortex, ATX is expressed by fibroblastic reticular cells and
generates LPA locally. LPA acts on lymphocytes via [LPA.sub.2] to
optimize T-cell movement, allowing the cells to navigate the highly
confined environment in the lymph node parenchyma.

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